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J. Li, J.A. Bonanno; The Effect of CFTR–siRNA and Vitamin C on Cl– and HCO3– Permeability in Bovine Corneal Endothelium Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2202.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Test the potential contribution of CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) to basal and stimulated Cl– and HCO3– permeability in bovine corneal endothelium cells (BCEC). Methods: Cultured BCEC were transfected with 20 nM CFTR targeted siRNA for 4 days, and protein was subjected to western blot analysis using anti–CFTR polyclonal antibody. Cl– permeability was measured with the halide–sensitive fluorescent dye MEQ. Steady–state HCO3– flux (difference in apical vs. basolateral pH after 6 hrs) was measured in the presence and absence of Vitamin C or forskolin, activators of CFTR. Results: Western blot analysis showed that CFTR expression was reduced ∼60% by CFTR–siRNA. Control cells treated with scrambled siRNA and perfused in the absence of Cl–, showed 0.112%/min MEQ quenching when apical Cl– was introduced. This was increased to 0.208%/min by 20 µM FSK. On the basolateral side MEQ quenching was increased from 0.034% to 0.10%/min with FSK. CFTR–siRNA treated cells showed 0.112%/min MEQ quenching on the apical side, but no increase (0.062%/min) with FSK. On the basolateral side, si–RNA treated cells showed 0.072% without and 0.090%/min quenching with FSK. BCEC steady–state HCO3– flux increased from +0.015 ± 0.0086 pH unit in control to +0.053 ± 0.0067 pH unit in 400 µM vitamin C. In 20 µM FSK stimulation, the HCO3– flux increased to +0.11 ± 0.025 pH unit. Conclusions: CFTR knockdown has no effect on unstimulated, but a significant effect on stimulated apical Cl– permeability. Vitamin C, which is normally in high concentrations in aqueous humor, can enhance the net HCO3– flux across the cultured bovine corneal endothelium.
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